ANALYTICAL
BIOCHEMISTRY
Application
66, 1oo- 103 ( 1975)
of Image
Intensifier
of Polyacrilamide VLADIMIR
for Autoradiography Gels
BRISGUNOV, BORIS REBENTISH, AND VLADIMIR DEBABOV
IRENE
GORDON,
Institute of Genetics and Selection of Industrial Microorganisms, Institute of Atomic Energy, Moscow, USSR Received August 26, 1974; accepted October 25, 1974 The new technique for autoradiography of polyacrilamide gels is described which utilizes image intensifier. Modified procedure reduces the exposure periods by factor of approximately 103-fold. The special advantage of the method is that it can be applied to wet gels which eliminates the procedure of gel drying.
Polyacrilamide gel electrophoresis has become widely used for separating mixtures of proteins (1) or of nucleic acids (2,3). In addition to its high resolution, the principal advantage of the method lies in the possibility of working with relatively small amounts of material (of the order of magnitude of micrograms or less). To make full use of polyacrilamide gels it is essential to be able to estimate radioactivity in macromolecular studies with fractional (longitudional) resolution comparable to that of the electrophoresis procedure. Autoradiography of the longitudional section of the column has been used to provide high resolution (4). Disadvantages of this procedure are too long exposure periods and difficulties of gel drying. During our studies of phage-induced proteins we came across all of these difficulties in practice. In our present communication the technique is described which allows radioactivity to be quickly registered in the longitudional section of the column with the use of image intensifier. This new technique reduces the exposure periods by factor of about 103. MATERIALS
AND
METHODS
(a) 14C-labeled lysates. 14C-labeled lysates were prepared as described by Laemmli (5). Twenty-milliliter cultures of E. coli B in M-9-glucose medium grown to 4 X IO* cells/ml were infected with T4 phages (wild type) at a multiplicity of 5. 14C-labeled amino acid mixture was added at the 2nd min after infection up to the final concentration of 20-30 pCi/ml. The infected cells were concentrated by the low-speed centrifugation at the 20th min after infection. The pellets were washed (M-9) and 100 Copy&M @ 1975 by Academic Press, Inc. AU rights of reproduction in any form reserved.
AUTORADIOGRAPHY
OF
POLYACRILINIDE
GELS
101
resuspended in 0.5 ml of sample buffer: 2% SDS, 5% ME, 10% glycerol in 0.0625 M Tris-HCI, pH 6,8. The whole lysate finally contained from 1 to l,5.107 cpmlml. The samples were counted on Mark-II liquid scintillation spectrometer. (b) Gel electrophoresis and autoradiography. The improved method of disc electrophoresis in acrylamide gels containing SDS was used (5). Gels were prepared from the stock solution of 30% acrilamide (by weight) and 0,8% of N,iV’-bis-a&amide. The separating gel contained in final concentrations: 0.375 M Tris-HCl, pH 8,8, and 0.1% SDS. The gels were polymerized chemically by the addition of 25 ~1 of tetramethylethylenediamine (Temed) and 0.3 ml of 10% ammonium persulfate solution/ 100 ml of gel solution. Ten-centimeter gels were prepared in glass tubes of a total length of 15 cm with inside diameter 6 mm. The stocking gels of length 1 cm contained 3% acrilamide, 0.125 M Tris-HCl, pH 6.8, and 0.1% SDS, and were also polymerized. The electrode buffer (pH 8.3) contained 0.025 M Tris and 0.192 M glycine and 0.1% SDS. The samples (0.1-0.2 ml) contained 0.001% bromphenol blue as the marker dye. The proteins were completely dissociated by immersing the samples for 5 min in boiling water bath. Electrophoresis was carried out with a current of 1,6 mA/gel until the bromphenol blue dye reached the bottom of the gel. The proteins were fixed in the gels with 50% trichloracetic acid for 10 min, and the gels were stained overnight with 0.25% Coomassie blue (Serva, GL) made up freshly in the mixture of methanol: acetic acid: water (5 : 1: 5). The gels were diffusionally destained by repeated washing in the 7% acetic acid. Autoradiophs of gels were prepared by the method of Fairbanks, Leventhal, and Reeder (4). Samples applied to the gel generally contained 5. 105-1 * 10” cpm, and 1 to 2 days exposure was usually sufficient. Tracing of appropriately exposed films and photofilms obtained from image intensifier were made with a IFO-451 microdensitometer. (c) Image intensifier technique. We used the electronic-optical fivestage image intensifier supplemented with thin window (30-40 mkm, mica). The fluorescent screen (ZnS, Ag, 20 mg/cm2) was deposited directly on a thin mica window. Operating field of device is a circle 40 mm in diameter; the resolution is better than 5 pair lines/mm. This intensifier has been originally designed for intensification of X-ray diffraction patterns (6), but the instrument is suited for registration of any soft radiation. At the present time some manufacturers produce analogous devices supplemented with the fiber optics faceplates. Autoradiography of the gels was performed by pressing of longitudional section of the gels to the input window of image intensifier. pparticles of 14C produce the luminescence of the ZnS screen. This luminescence is amplified by image intensifier and registered by 35-mm
102
BRISGLJNOV
ET AL.
FIG. I. Autoradiographs of W-labeled phage-induced proteins made (a) by routine method (exposure time 24 hr); (b) with the use of image intensifier (exposure time 2 mm). The total radioactivity loaded to each gel was about 1,2. 10s counts/100 sec. Fifty discrete bands were resolved along the whole column.
FIG. 2. Densitometer tracing of the autoradiographs shown in Fig. la of the autoradiograph made by routine method; (b) of the autoradiograph made by the use of image intensifier.
AUTORADIOGRAPHY
OF
POLYACRILINIDE
103
GELS
mirror camera. The exposure time has to be determined for each series of gels and depends on the gain of image intensifier and effective aperture ratio for photo objective. In practice the time of exposure varies from one to 120 sec. RESULTS
In Fig. 1 the autoradiographs are compared obtained by the method of Fairbanks et al. (la) and by the method with the use of image intensifier (lb). The application of image intensifier technique allows 720-fold decrease of exposure period. The quality of the autoradiographs obtained by image intensifier method is influenced by the granularity (Fig. lb). This disadvantage can be eliminated by the tracing of the films, hence two densitoradiographs obtained by these two methods are practically indiscernible (Fig. 2a and b). Under high gain of image intensifier single p-particle scintillation becomes easily detectable by photographic emulsion, so that count of pparticle traces permits the reduction of the errors due to recording of X-ray film intensities. The short exposure periods permit us to work with wet gels which eliminates time of consuming (up to 90 hr) and laborious procedures of gel drying. The possibility of autoradiography of samples with low radioactivity is limited by the dark background of image intensifier. It should be stressed that low dark background is specially important when dealing with soft radiation (e.g., 3H). REFERENCES I. 2. 3. 4.
Gel electrophoresis, Ann. N.Y. Acad. Sci. Ark/e 2. Loening, U. I. (1967) Biochem. J. 102, 251. Peacock, A. C., and Dingman, C. W. (1968) Biochemistry 7, Fairbanks, J., Jr., Leventhal, C., and Reeder, R. H. (1965) Commun.
20,
668. Biochem.
Biophys.
Res.
393.
5. Laemmli, U. F. (1970) Nature (London) 227, 680. 6. Brizgunov. V. A.. Butslov, M. M., Mokulsky, M. A.. (1969)
DAN
USSR
185,
782.
BIOCHEMISTRY
Application
66, 1oo- 103 ( 1975)
of Image
Intensifier
of Polyacrilamide VLADIMIR
for Autoradiography Gels
BRISGUNOV, BORIS REBENTISH, AND VLADIMIR DEBABOV
IRENE
GORDON,
Institute of Genetics and Selection of Industrial Microorganisms, Institute of Atomic Energy, Moscow, USSR Received August 26, 1974; accepted October 25, 1974 The new technique for autoradiography of polyacrilamide gels is described which utilizes image intensifier. Modified procedure reduces the exposure periods by factor of approximately 103-fold. The special advantage of the method is that it can be applied to wet gels which eliminates the procedure of gel drying.
Polyacrilamide gel electrophoresis has become widely used for separating mixtures of proteins (1) or of nucleic acids (2,3). In addition to its high resolution, the principal advantage of the method lies in the possibility of working with relatively small amounts of material (of the order of magnitude of micrograms or less). To make full use of polyacrilamide gels it is essential to be able to estimate radioactivity in macromolecular studies with fractional (longitudional) resolution comparable to that of the electrophoresis procedure. Autoradiography of the longitudional section of the column has been used to provide high resolution (4). Disadvantages of this procedure are too long exposure periods and difficulties of gel drying. During our studies of phage-induced proteins we came across all of these difficulties in practice. In our present communication the technique is described which allows radioactivity to be quickly registered in the longitudional section of the column with the use of image intensifier. This new technique reduces the exposure periods by factor of about 103. MATERIALS
AND
METHODS
(a) 14C-labeled lysates. 14C-labeled lysates were prepared as described by Laemmli (5). Twenty-milliliter cultures of E. coli B in M-9-glucose medium grown to 4 X IO* cells/ml were infected with T4 phages (wild type) at a multiplicity of 5. 14C-labeled amino acid mixture was added at the 2nd min after infection up to the final concentration of 20-30 pCi/ml. The infected cells were concentrated by the low-speed centrifugation at the 20th min after infection. The pellets were washed (M-9) and 100 Copy&M @ 1975 by Academic Press, Inc. AU rights of reproduction in any form reserved.
AUTORADIOGRAPHY
OF
POLYACRILINIDE
GELS
101
resuspended in 0.5 ml of sample buffer: 2% SDS, 5% ME, 10% glycerol in 0.0625 M Tris-HCI, pH 6,8. The whole lysate finally contained from 1 to l,5.107 cpmlml. The samples were counted on Mark-II liquid scintillation spectrometer. (b) Gel electrophoresis and autoradiography. The improved method of disc electrophoresis in acrylamide gels containing SDS was used (5). Gels were prepared from the stock solution of 30% acrilamide (by weight) and 0,8% of N,iV’-bis-a&amide. The separating gel contained in final concentrations: 0.375 M Tris-HCl, pH 8,8, and 0.1% SDS. The gels were polymerized chemically by the addition of 25 ~1 of tetramethylethylenediamine (Temed) and 0.3 ml of 10% ammonium persulfate solution/ 100 ml of gel solution. Ten-centimeter gels were prepared in glass tubes of a total length of 15 cm with inside diameter 6 mm. The stocking gels of length 1 cm contained 3% acrilamide, 0.125 M Tris-HCl, pH 6.8, and 0.1% SDS, and were also polymerized. The electrode buffer (pH 8.3) contained 0.025 M Tris and 0.192 M glycine and 0.1% SDS. The samples (0.1-0.2 ml) contained 0.001% bromphenol blue as the marker dye. The proteins were completely dissociated by immersing the samples for 5 min in boiling water bath. Electrophoresis was carried out with a current of 1,6 mA/gel until the bromphenol blue dye reached the bottom of the gel. The proteins were fixed in the gels with 50% trichloracetic acid for 10 min, and the gels were stained overnight with 0.25% Coomassie blue (Serva, GL) made up freshly in the mixture of methanol: acetic acid: water (5 : 1: 5). The gels were diffusionally destained by repeated washing in the 7% acetic acid. Autoradiophs of gels were prepared by the method of Fairbanks, Leventhal, and Reeder (4). Samples applied to the gel generally contained 5. 105-1 * 10” cpm, and 1 to 2 days exposure was usually sufficient. Tracing of appropriately exposed films and photofilms obtained from image intensifier were made with a IFO-451 microdensitometer. (c) Image intensifier technique. We used the electronic-optical fivestage image intensifier supplemented with thin window (30-40 mkm, mica). The fluorescent screen (ZnS, Ag, 20 mg/cm2) was deposited directly on a thin mica window. Operating field of device is a circle 40 mm in diameter; the resolution is better than 5 pair lines/mm. This intensifier has been originally designed for intensification of X-ray diffraction patterns (6), but the instrument is suited for registration of any soft radiation. At the present time some manufacturers produce analogous devices supplemented with the fiber optics faceplates. Autoradiography of the gels was performed by pressing of longitudional section of the gels to the input window of image intensifier. pparticles of 14C produce the luminescence of the ZnS screen. This luminescence is amplified by image intensifier and registered by 35-mm
102
BRISGLJNOV
ET AL.
FIG. I. Autoradiographs of W-labeled phage-induced proteins made (a) by routine method (exposure time 24 hr); (b) with the use of image intensifier (exposure time 2 mm). The total radioactivity loaded to each gel was about 1,2. 10s counts/100 sec. Fifty discrete bands were resolved along the whole column.
FIG. 2. Densitometer tracing of the autoradiographs shown in Fig. la of the autoradiograph made by routine method; (b) of the autoradiograph made by the use of image intensifier.
AUTORADIOGRAPHY
OF
POLYACRILINIDE
103
GELS
mirror camera. The exposure time has to be determined for each series of gels and depends on the gain of image intensifier and effective aperture ratio for photo objective. In practice the time of exposure varies from one to 120 sec. RESULTS
In Fig. 1 the autoradiographs are compared obtained by the method of Fairbanks et al. (la) and by the method with the use of image intensifier (lb). The application of image intensifier technique allows 720-fold decrease of exposure period. The quality of the autoradiographs obtained by image intensifier method is influenced by the granularity (Fig. lb). This disadvantage can be eliminated by the tracing of the films, hence two densitoradiographs obtained by these two methods are practically indiscernible (Fig. 2a and b). Under high gain of image intensifier single p-particle scintillation becomes easily detectable by photographic emulsion, so that count of pparticle traces permits the reduction of the errors due to recording of X-ray film intensities. The short exposure periods permit us to work with wet gels which eliminates time of consuming (up to 90 hr) and laborious procedures of gel drying. The possibility of autoradiography of samples with low radioactivity is limited by the dark background of image intensifier. It should be stressed that low dark background is specially important when dealing with soft radiation (e.g., 3H). REFERENCES I. 2. 3. 4.
Gel electrophoresis, Ann. N.Y. Acad. Sci. Ark/e 2. Loening, U. I. (1967) Biochem. J. 102, 251. Peacock, A. C., and Dingman, C. W. (1968) Biochemistry 7, Fairbanks, J., Jr., Leventhal, C., and Reeder, R. H. (1965) Commun.
20,
668. Biochem.
Biophys.
Res.
393.
5. Laemmli, U. F. (1970) Nature (London) 227, 680. 6. Brizgunov. V. A.. Butslov, M. M., Mokulsky, M. A.. (1969)
DAN
USSR
185,
782.
